Cronobacter spp. (previously Enterobacter sakazakii) invade and translocate across both cultured human intestinal epithelial cells and human brain microvascular endothelial cells Chandrakant P. Giri a , Kensuke Shima a , Ben D. Tall b , Sherill Curtis b , Venugopal Sathyamoorthy b , Brock Hanisch a , Kwang S. Kim c , Dennis J. Kopecko a, * a Laboratory of Enteric and Sexually Transmitted Diseases, DBPAP, OVRR, Center for Biologics Evaluation and Research, Food & Drug Administration, Bethesda, MD 20892, USA b Division of Virulence Assessment, Center for Food Safety and Applied Nutrition, Food & Drug Administration, Laurel, MD 20708, USA c Pediatric Infectious Diseases, School of Medicine, Johns Hopkins University, Baltimore, MD 2128, USA article info Article history: Received 3 January 2011 Received in revised form 7 October 2011 Accepted 10 October 2011 Available online xxx Keywords: Transcytosis Meningitis Cronobacter sakazakii Bloodebrain barrier CNS abstract The mechanism of Cronobacter pathogenesis in neonatal meningitis and potential virulence factors (aside from host cell invasion ability) remain largely unknown. To ascertain whether Cronobacter can invade and transcytose across intestinal epithelial cells, enter into the blood stream and then transcytose across the blood-brain-barrier, we have utilized human intestinal INT407 and Caco-2 cells and brain micro- vascular endothelial cell (HBMEC) monolayers on Transwell filters as experimental model systems. Our data indicate a wide range of heterogeneity with respect to invasion efficiency among twenty-three Cronobacter isolates screened. For selected isolates, we observed significant levels of transcytosis for Cronobacter sakazakii across tight monolayers of both Caco-2 and HBMEC, mimicking in vivo ability to cross the intestine as well as the blood brain barrier, and at a frequency equivalent to that of a control meningitis-causing Escherichia coli K1 strain. Finally, EM analysis demonstrated intracellular Cronobacter bacteria within host vacuoles in HBMEC, as well as transcytosed bacteria at the basolateral surface. These data reveal that certain Cronobacter isolates can invade and translocate across both cultured human intestinal epithelial cells and HBMEC, thus demonstrating a potential path for neonatal infections of the central nervous system (CNS) following oral ingestion. Published by Elsevier Ltd. 1. Introduction Enterobacter spp. are motile, rod-shaped, non-spore forming, Gram-negative, facultatively anaerobic members of the family Enterobacteriaceae. Although considered normal inhabitants of the gastrointestinal tract, Enterobacter are common contaminants of inanimate hospital surfaces and, as such, are responsible for w10% of nosocomial infections, particularly wound infections, bacteremia, and pneumonia [1]. Enterobacter sakazakii is uniquely associated with infections in neonates and can cause necrotizing enterocolitis, bacteremia, and meningitis, with reported mortality rates of 40e80%; even the survivors often develop chronic neurological and developmental disorders [2e6]. There is considerable diversity with respect to both genotypic and phenotypic characteristics among different isolates of E. sakazakii. Accordingly, Iversen and coworkers [7e9], recently proposed a taxonomic reclassification of E. sakazakii, to Cronobacter spp., because E. sakazakii isolates are both genetically distinct from other Enterobacter spp. and represent at least six different species; recently, Joseph et al. [10] added two new species to this genus. While this pathogen has been detected in a wide variety of foods [5,11], powdered infant formula remains primarily linked to outbreaks of neonatal meningitis. The mechanism of pathogenicity and expression of potential virulence factors of Cronobacter spp. remain largely unknown. To date, the few reports describing putative virulence factors are: by Pagotto et al. [12], who evaluated various E. sakazakii isolates for production of an enterotoxin using the suckling mouse assay; by Kothary et al. [13] who described * Corresponding author. Laboratory of Enteric and Sexually Transmitted Diseases, FDA, Center for Biologics Evaluation & Research, HFM-440, 29 Lincoln Drive, NIH Campus Bldg 29/420, Bethesda, MD 20892, USA. Tel.: þ1 301 496 1893; fax: þ1 301 402 8701. E-mail addresses: chandrakant.giri@fda.hhs.gov (C.P. Giri), kensuke.shima@uk- sh.de (K. Shima), ben.tall@fda.hhs.gov (B.D. Tall), sherill.curtis@fda.hhs.gov (S. Curtis), venugopal.sathyamoorthy@fda.hhs.gov (V. Sathyamoorthy), brock. hanisch@fda.hhs.gov (B. Hanisch), kwangkim@jhmi.edu (K.S. Kim), dennis. kopecko@fda.hhs.gov (D.J. Kopecko). Contents lists available at SciVerse ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath 0882-4010/$ e see front matter Published by Elsevier Ltd. doi:10.1016/j.micpath.2011.10.003 Microbial Pathogenesis xxx (2011) 1e8 Please cite this article in press as: Giri CP, et al., Cronobacter spp. (previously Enterobacter sakazakii) invade and translocate across both cultured human intestinal epithelial cells and human brain microvascular endothelial cells, Microbial Pathogenesis (2011), doi:10.1016/ j.micpath.2011.10.003